FADS genetic variants and ω-6 polyunsaturated fatty acid metabolism in a homogeneous island population

Long-chain polyunsaturated fatty acids (PUFA) orchestrate immunity and inflammation through their capacity to be converted to potent inflammatory mediators. We assessed associations of FADS gene cluster polymorphisms and fasting serum PUFA concentrations in a fully ascertained, geographically isolated founder population of European descent. Concentrations of 22 PUFAs were determined by gas chromatography, of which ten fatty acids and five ratios defining FADS1 and FADS2 activity were tested for genetic association against 16 single nucleotide polymorphisms (SNP) in 224 individuals. A cluster of SNPs in tight linkage disequilibrium in the FADS1 gene (rs174537, rs174545, rs174546, rs174553, rs174556, rs174561, rs174568, and rs99780) were strongly associated with arachidonic acid (AA) (P = 5.8 × 10⁻⁷ – 1.7 × 10⁻⁸) among other PUFAs, but the strongest associations were with the ratio measuring FADS1 activity in the ω-6 series (P = 2.11 × 10⁻¹³ – 1.8 × 10⁻²⁰). The minor allele across all SNPs was consistently associated with decreased ω-6 PUFAs, with the exception of dihomo-γ-linoleic acid (DHGLA), where the minor allele was consistently associated with increased levels. Our findings in a geographically isolated population with a homogenous dietary environment suggest that variants in the Δ-5 desaturase enzymatic step likely regulate the efficiency of conversion of medium-chain PUFAs to potentially inflammatory PUFAs, such as AA.     

Phosphorylation of p22phox on Threonine 147 Enhances NADPH Oxidase Activity by Promoting p47phox Binding

NADPH oxidase comprises both cytosolic and membrane-bound subunits, which, when assembled and activated, initiate the transfer of electrons from NADPH to molecular oxygen to form superoxide. This activity, known as the respiratory burst, is extremely important in the innate immune response as indicated by the disorder chronic granulomatous disease. The regulation of this enzyme complex involves protein-protein and protein-lipid interactions as well as phosphorylation events. Previously, our laboratory demonstrated that the small membrane subunit of the oxidase complex, p22^phox, is phosphorylated in neutrophils and that its phosphorylation correlates with NADPH oxidase activity. In this study, we utilized site-directed mutagenesis in a Chinese hamster ovarian cell system to determine the phosphorylation sites within p22^phox. We also explored the mechanism by which p22^phox phosphorylation affects NADPH oxidase activity. We found that mutation of threonine 147 to alanine inhibited superoxide production in vivo by more than 70%. This mutation also blocked phosphorylation of p22^phox in vitro by both protein kinase C-α and -δ. Moreover, this mutation blocked the p22^phox-p47^phox interaction in intact cells. When phosphorylation was mimicked in vivo through mutation of Thr-147 to an aspartyl residue, NADPH oxidase activity was recovered, and the p22^phox-p47^phox interaction in the membrane was restored. Maturation of gp91^phox was not affected by the alanine mutation, and phosphorylation of the cytosolic component p47^phox still occurred. This study directly implicates threonine 147 of p22^phox as a critical residue for efficient NADPH oxidase complex formation and resultant enzyme activity.     

A method for C-terminal sequence analysis in the proteomic era (proteins cleaved with cyanogen bromide)

There is growing interest in the overall study of post-translational modifications (PTMs) of proteins. Beside phosphorylation and glycosylation, truncations of the nascent polypeptide chain at the N or C termini are by far the most common types of PTMs found in proteins. However, little attention has been paid to the development of approaches that allow a systematic analysis of these proteolytic processing events. Here we present a protocol that allows the identification of the C-terminal sequences of proteins. A peptide mixture is generated by cleavage of the protein with cyanogen bromide and is incubated with carboxypeptidase Y. The enzyme is only able to act on the C-terminal fragment, because this is the only peptide without a homoserine lactone residue at its C terminus. The resulting fragments, forming a peptide ladder, are analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The entire protocol, including the CNBr cleavage, takes 21 h and can be applied to proteins purified either by SDS-PAGE or by 2D PAGE or in solution.     

Interstitial cells of Cajal in the urethra

The smooth muscle layer of the urethra generates spontaneous myogenic tone that is thought to make a major contribution to urinary continence. The mechanisms underlying generation of tone remain unclear, however recent studies from our laboratory highlighted a role for a specialised population of pacemaker cells which we originally referred to as interstitial cells (IC) and now term ICC. Urethra ICC possess an electrical pacemaker mechanism characterised by rhythmic activation of Ca(2+)-activated Cl(-) channels leading to spontaneous transient inward currents (STICs) under voltage clamp and spontaneous transient depolarisations (STDs) under current clamp conditions. Both STICS and STDs are now known to be associated with spontaneous Ca(2+) oscillations that result from a complex interplay between release of Ca(2+) from intracellular stores and Ca(2+) influx across the plasma membrane. In this review we will consider some of the precise mechanisms involved in the generation of pacemaker activity and discuss how these are modulated by excitatory and inhibitory neurotransmitters.     

The C-terminal region but not the Arg-X-Pro repeat of Epstein-Barr virus protein EB2 is required for its effect on RNA splicing and transport

The Epstein-Barr virus BMLF1 gene product EB2 has been shown to efficiently transform immortalized Rat1 and NIH 3T3 cells, to bind RNA, and to shuttle from the nucleus to the cytoplasm. In transient-expression assays EB2 seems to affect mRNA nuclear export of intronless RNAs and pre-mRNA 3' processing, but no direct proof of EB2 being involved in RNA processing and transport has been provided, and no specific functional domain of EB2 has been mapped. Here we significantly extend these findings and directly demonstrate that (i) EB2 inhibits the cytoplasmic accumulation of mRNAs, but only if they are generated from precursors containing weak (cryptic) 5' splice sites, (ii) EB2 has no effect on the cytoplasmic accumulation of mRNA generated from precursors containing constitutive splice sites, and (iii) EB2 has no effect on the 3' processing of precursor RNAs containing canonical and noncanonical cleavage-polyadenylation signals. We also show that in the presence of EB2, intron-containing and intronless RNAs accumulate in the cytoplasm. EB2 contains an Arg-X-Pro tripeptide repeated eight times, similar to that described as an RNA-binding domain in the herpes simplex virus type 1 protein US11. As glutathione S-transferase fusion proteins, both EB2 and the Arg-X-Pro repeat bound RNA in vitro. However, by using EB2 deletion mutants, we demonstrated that the effect of EB2 on splicing and RNA transport requires the C-terminal half of the protein but not the Arg-X-Pro repeat.     

Truncated beta-amyloid peptide species in pre-clinical Alzheimer's disease as new targets for the vaccination approach

Vaccination against human beta-amyloid peptide (A beta) has been shown to remove the amyloid burden produced in transgenic mice overexpressing the mutated human amyloid precursor protein (APP) gene. For human beings, the efficiency of this therapeutic strategy has to take into account the specificities of human amyloid, especially at the early stages of 'sporadic' Alzheimer's disease (AD). A beta 40/42 were previously quantified in tissues from our well-established brain bank, including non-demented individuals with both mild amyloid and tau pathologies, hence corresponding to the earliest stages of Alzheimer pathology. Herein, we have adapted a proteomic method combined with western blotting and mass spectrometry for the characterization of insoluble A beta extracted in pure-formic acid. We demonstrated that amino-truncated A beta species represented more than 60% of all A beta species, not only in full blown AD, but also, and more interestingly, at the earliest stage of Alzheimer pathology. At this stage, A beta oligomers were exclusively made of A beta-42 species, most of them being amino-truncated. Thus, our results strongly suggest that amino-truncated A beta-42 species are instrumental in the amyloidosis process. In conclusion, a vaccine specifically targeting these pathological amino-truncated species of A beta-42 are likely to be doubly beneficial, by inducing the production of specific antibodies against pathological A beta products that are, in addition, involved in the early and basic mechanisms of amyloidosis in humans.